一种来源于Burkholderia phytofirmans PsJN的ω-转氨酶的表达纯化及性质分析  

Expression and characterization of a novel ω-transaminase from Burkholderia phytofirmans Ps JN

作　　者：杜允成[1,2] 董文玥[1] 姜进举[1,2] 陈启佳[1,2] 冯进辉[1] 吴洽庆[1] 朱敦明[1] 

机构地区：[1]中国科学院天津工业生物技术研究所工业酶国家工程实验室,天津300308 [2]中国科学院大学,北京100049

出　　处：《生物工程学报》2016年第7期912-926,共15页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(No.2011CB710800)资助~~

摘　　要：ω-转氨酶(ω-transaminase)可以通过手性拆分和不对称合成的催化反应来获得光学纯的手性胺类化合物和非天然氨基酸,在医药中间体合成中是一种重要的生物催化剂。采用基因挖掘技术,在基因组数据库中获得一个来自伯克氏菌Burkholderia phytofirmans Ps JN的ω-转氨酶基因(hbp),将该基因在大肠杆菌BL21(DE3)中克隆、表达,利用镍柱亲和层析将该酶(HBP)进行纯化并研究了其酶学性质和底物谱。结果表明,以β-苯丙氨酸(β-Phe)为氨基供体、丙酮酸为氨基受体,HBP具有较高的活力(33.80 U/mg)和立体选择性;其最适温度为40℃左右,最适p H在8.0–8.5之间。研究过程中,建立了一种简便快捷的紫外吸收法来检测β-Phe的脱氨反应,证明了该反应的热力学平衡性质。底物谱研究表明HBP可以以β-Phe及其衍生物为氨基供体。结果表明HBP能够有效地手性拆分rac-β-Phe及其衍生物,转化率在50%左右,ee>99%。Production of chiral amines and unnatural amino-acid using ω-transaminase can be achieved by kinetic resolution and asymmetric synthesis, thus ω-transaminase is of great importance in the synthesis of pharmaceutical intermediates. By genomic data mining, a putative ω-transaminase gene hbp was found in Burkholderia phytofirmans Ps JN. The gene was cloned and over-expressed in Escherichia coli BL21（DE3）. The recombinant enzyme（HBP） was purified by Ni-NTA column and its catalytic properties and substrate profile were studied. HBP showed high relative activity（33.80 U/mg） and enantioselectivity toward β-phenylalanine（β-Phe）. The optimal reaction temperature and p H were 40 ℃ and 8.0–8.5, respectively. We also established a simpler and more effective method to detect the deamination reaction of β-Phe by UV absorption method using microplate reader, and demonstrated the thermodynamic property of this reaction. The substrate profiling showed that HBP was specific to β-Phe and its derivatives as the amino donor. HBP catalyzed the resolution of rac-β-Phe and its derivatives, the products（R）-amino acids were obtained with about 50% conversions and 99% ee.

关 键 词：ω-转氨酶 伯克氏菌 芳香族β-氨基酸 手性拆分 

分 类 号：Q936[生物学—微生物学] Q78